Terry Wood

The separation of isomeric pentose phosphates from each other and the preparation of D-xylulose 5-phosphate and D-ribulose 5-phosphate by column chromatography.

Abstract The binding of the 5-phosphates of ribose, arabinose, ribulose, and xylulose to a dihydroxyboryl-substituted cellulose column was investigated under various conditions of pH, salt content, and the presence of ethanol. Ribose 5-phosphate was bound to the column at pH 8.1 or above at a high ionic strength when hydrazine was absent but not when it was present. Xylulose 5-phosphate was bound with hydrazine present while ribulose 5-phosphate only bound strongly when both hydrazine and ethanol were present. The structural basis for this behavior is discussed. Procedures are described for isolating ribulose 5-phosphate from an isomerase equilibrium mixture with ribose 5-phosphate, and for isolating xylulose 5-phosphate from a mixture containing ribulose and ribose 5-phosphates.

The preparation of transketolase free from d-ribulose-5-phosphate 3-epimerase

A procedure for the purification from Candida utilis of transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) free from D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) was developed using acetone precipitation, elution from DEAE-cellulose, adsorption of epimerase by thiopropyl-Sepharose, and chromatography on D-ribose 5-phosphate-Sepharose and DEAE--Sephadex. The final product had a specific activity of 43 units/mg, a transketolase/epimerase activity ratio greater than 53 000 to 1, an apparent Km for D-xylulose 5-phosphate and D-ribose 5-phosphate of 77 and 430 microM, respectively, and ran as a single band using electrophoresis on polyacrylamide gel. It was inhibited by D-arabinose 5-phosphate and D-glucose 6-phosphate. During the purification by column chromatography, multiple forms of the enzyme were detected by gel electrophoresis but these gradually disappeared as the enzyme was further purified.

Complex formation between transketolase, transaldolase, and glyceraldehyde phosphate dehydrogenase

1. 1. The existence of complexes between transketolase, transaldolase, and glyceraldehyde phosphate dehydrogenase from Candida utilis was investigated. 2. 2. Evidence of complex formation was obtained by gel filtration, chromatography on DEAE-Sephadex, and gel electrophoresis. 3. 3. The complexes were fairly stable at pH 6.0 but dissociated slowly in the pH range 7.4–8.5 with a concomitant increase in the activities of transaldolase and glyceraldehyde phosphate dehydrogenase. 4. 4. The partial purification of glyceraldehyde phosphate dehydrogenase from Candida utilis is described. 5. 5. Incubation of the above preparation with pure transketolase and transaldolase at pH 6.4 led to the formation of binary complexes of transketolase with transaldolase, of transketolase with glyceraldehyde phosphate dehydrogenase, and of a ternary complex. 6. 6. The function of the ternary complex was suggested to be the coupled conversion of 1,3-bisphosphoglycerate and fructose 6-phosphate to pentose phosphate and experimental evidence was obtained for the feasibility of the reaction.

The electrophoresis and detection of transketolase.

Abstract After electrophoresis impure transketolase preparations stained readily with an activity stain based on a published method, but pure preparations gave no reaction. The bands first obtained were due to the presence of (i) a transketolase- d -glyceraldehyde-3-phosphate dehydrogenase complex, (ii) alcohol dehydrogenases, and (iii) a zone of high local pH at the buffer front. Lyophilization of the reagents eliminated artifacts due to alcohol dehydrogenase. An external starch indicator gel was developed that gave colored bands with both pure and impure transketolase preparations and a similar indicator gel was developed for transaldolase.